G-NOME^® DNA Isolation Kit
High Molecular Weight Genomic DNA Isolation Kit
Revision No. 2010-999-5H01W

The kit is shipped at ambient temperature. However, upon arrival, store Protease Mixx at 4°C. A few days at ambient temperature does not affect activity of this reagent. A precipitate (often appears fungus-like) is sometimes evident after storage. Remove through centrifugation. Protease activity is not affected.

The G NOME Kit is used to quickly and efficiently isolate high molecular weight genomic DNA from cells and tissues of any type, including bacteria, yeast, plant, and animal cells. Each preparation with the G NOME Kit yields up to 100 mg of genomic DNA. The DNA isolated by the G NOME procedure is suitable for restriction enzyme digestion or PCR amplification in as little as 1 hour after cell lysis.

The G NOME Kit is available in 3 sizes to suit your needs: 10, 25, and 100 preparations/kit. The G NOME Kit utilizes RNase Mixx to eliminate RNA immediately after lysis, and Protease Mixx to rapidly degrade cellular proteins. This is followed by a proprietary "salting-out" technique which precludes the need for phenol, chloroform, or other organic extractions. The DNA can either be spooled, removed with forceps, or centrifuged and dissolved in TE.

The G NOME Kit contains the reagents to homogenize cells or tissue and purify high molecular weight DNA. Ethanol is the only reagent not supplied in the kit. Lysing enzymes for yeast or bacterial cells are available separately. Contact BIO 101 for information.

1. Bring cells*/tissues to a final volume of 1.85 ml in Cell Suspension Solution. (Use a 15 ml clear plastic tube for efficient mixing). Mix until the suspension appears homogeneous.
2. Add 50 µl of RNase Mixx; mix thoroughly.
3. Add 100 µl of Cell Lysis/Denaturing Solution**; mix well.
4. Incubate at 55°C for 15 minutes.
5. Add 25 µl Protease Mixx; mix thoroughly.
6. Incubate at 55°C for 30 to 120 minutes. (The longer time will result in minimal protein carry-over and will also allow for substantial reduction in residual protease activity.)
7. Add 500 µl "Salt-Out" Mixture; mix gently yet thoroughly. Divide sample into 1.5 ml tubes. Refrigerate at 4°C for 10 minutes.
8. Spin for 10 minutes at maximum speed in a microcentrifuge (at least 10,000 x g). Carefully collect the supernatant, avoiding the pellet. If a precipitate remains in the supernatant, cool on ice and spin again until it is clear. Pool the supernatants in a 15 ml (or larger) clear plastic tube.
9. To this supernatant, add 2 ml TE (10mM Tris pH 7.5, 1mM EDTA) buffer and mix. Then add 8 ml of 100% ethanol. If spooling the DNA, add the ethanol slowly and spool the DNA at the interphase with a clean glass rod. If centrifuging the DNA, add the ethanol and gently mix the solution by inverting the tube. Spin for 15 minutes at 1000-1500 xg. Eliminate the excess ethanol by blotting or air drying the DNA.
10. Dissolve the genomic DNA in TE buffer.

** If a precipitate forms in the stock solution, heat to 60°C to dissolve before using.

The G NOME Kit is designed to yield 100 mg of genomic DNA based on the following amounts of starting material and cellular prelysis methods. The protocol will accommodate a variety of cell preparation methods with the following provided as a guideline.

Bacteria
5 x 10^-15g DNA/E. coli, K12
Inoculate a single colony of bacteria into 5ml of growth media. Grow overnight at 37°C with shaking and pellet cells. The wet pellet weight should be approximately 20-50 mg. Continue with step 1 of the general G NOME protocol.

Plant Tissue
5.1 x 10^-12g DNA/tobacco plant cell Place 250 mg of fresh tissue in a microcentrifuge tube and immerse in liquid nitrogen to freeze. Grind the tissue to a fine powder with a small conical-shaped pestle, adding liquid nitrogen as necessary to keep the tissue frozen. Add 500 µl Cell Suspension Solution and continue to grind tissue with the tube on ice until it appears homogeneous. Add an additional 1.35 ml Cell Suspension Solution. Continue with step 2 of the G NOME protocol.

Blood Cells, see pg 63.

Mammalian Tissue
3.5 (human), 3.2 (rat) x 10^-12g DNA/cell
Completely homogenize enough tissue to yield approximately 2-5 x 10⁷ cells in 1 ml of Cell Suspension Solution. This will vary between 50 to 250 mg depending upon the density of cells in the type of tissue. Continue with step 1 of the G NOME protocol.

Yeast Cells
1.6 x 10^-14g DNA/cell, S. cerevisiae. Lysing reagents for yeast cells are available separately. See pg 61 for information on the Yeast Cell Lysis Kit.